Hepatitis B is a viral disease transmitted parenterally by contaminated material such as blood and blood products, contaminated needles, sexually and vertically from infected or carrier mothers to their offspring. In those areas of the world where the disease is common vertical transmission at an early age results in a high proportion of infected individuals becoming chronic carriers of hepatitis B. It is estimated by the World Health Organization that more than 2 billion people have been infected worldwide, with about 4 million acute cases per year, 1 million deaths per year, and 350-400 million chronic carriers. Approximately 25% of carriers die from chronic hepatitis, cirrhosis, or liver cancer and nearly 75% of chronic carriers are Asian. Hepatitis B virus is the second most significant carcinogen behind tobacco, causing from 60% to 80% of all primary liver cancer. HBV is 100 times more contagious than HIV.
Hepatitis B viral infections are a continuing medical problem because, like any rapidly-replicating infectious agent, there are continuing mutations that help some sub-populations of HBV become resistant to current treatment regimens. At the present time there are no effective therapeutic agents for treating humans infected with HBV infections which result in seroconversion to the virus in the body, or which effect a 90% reduction of antigen, compared to baseline numbers before treatment, in persons suffering from a hepatitis B viral infection. Currently the recommended therapies for chronic HBV infection by the American Association for the Study of Liver Diseases (AASLD) and the European Association for the Study of the Liver (EASL) include interferon alpha (INFα), pegylated interferon alpha-2a (Peg-IFN2a), entecavir, and tenofovir. However, typical interferon therapy is 48-weeks and results in serious and unpleasant side effects, and HBeAg seroconversion, 24 weeks after therapy has ceased, ranges from only 27-36%. Seroconversion of HBsAg is even lower—only 3% observed immediately after treatment ceases, with an increase to upwards of 12% after 5 years.
The nucleoside and nucleotide therapies entecavir and tenofovir are successful at reducing viral load, but the rates of HBeAg seroconversion and HBsAg loss are even lower than those obtained using IFNα therapy. Other similar therapies, including lamivudine (3TC), telbivudine (LdT), and adefovir are also used, but for nucleoside/nucleotide therapies in general, the emergence of resistance limits therapeutic efficacy.
Thus, there is a need in the art to discover and develop new anti-viral therapies. More particularly, there is a need for new anti-HBV therapies capable of increasing HBeAg and HBsAg seroconversion rates. These serum markers are indicative of immunological control of HBV infection and leads to an improved prognosis, i.e. prevention of liver disease and progression to cirrhosis, prevention of liver failure, prevention of hepatocellular cancer (HCC), prevention of liver disease—related transplantation, and prevention of death.
Recent clinical research has found a correlation between seroconversion and reductions in HBeAg (Fried et al (2008) Hepatology 47:428) and reductions in HBsAg (Moucari et al (2009) Hepatology 49:1151). Reductions in antigen levels may have allowed immunological control of HBV infection because high levels of antigens are thought to induce immunological tolerance. Current nucleoside therapies for HBV are capable of dramatic reductions in serum levels of HBV but have little impact on HBeAg and HBsAg levels. Antisense therapy differs from nucleoside therapy in that it can directly target the transcripts for the antigens and thereby reduce serum HBeAg and HBsAg levels. Because of the multiple, overlapping transcripts produced upon HBV infection, there is also an opportunity for a single antisense oligomer to reduce HBV DNA in addition to both HBeAg and HBsAg.
Antisense therapy is a form of treatment for genetic disorders or infections. When the genetic sequence of a particular gene is known to be causative of a particular disease, whether the gene is an original mammalian gene, an oncogene, or a gene from an infective organism, such as a gene from a bacterial species, a gene from a fungus, a gene from a parasite or a gene from a virus, it is possible to synthesize a strand of nucleic acid (DNA, RNA or a chemical analogue) that will bind to the messenger RNA (mRNA) produced by that gene and inactivate it, effectively turning that gene “off”. This is because mRNA must be single stranded for it to be translated. Alternatively, the strand might be targeted to bind a splicing site on pre-mRNA and modify the exon content of an mRNA[1].
A DNA single strand sequence is often called the sense strand (or positive (+) sense strand) if an RNA version having the same sequence (except U in RNA for T in DNA) is translated or translatable into protein, and the complementary strand is called the antisense strand (or negative (−) sense strand).
Some regions within a double strand of DNA code for genes, which are usually instructions specifying the order of amino acids in an expressed, or translated, protein, together with regulatory sequences, splicing sites, noncoding introns and other regions. For a cell to express the protein coded by the DNA, one strand of the DNA serves as a template for the synthesis of a complementary strand of RNA. The template DNA strand is called the transcribed strand and its sequence is antisense, or complementary, to the mRNA transcript, which has the same sequence as the sense sequence of the original double-stranded DNA. Because the DNA is double-stranded, the strand complementary to the antisense sequence is called the non-transcribed strand, or sense strand, and has the same sequence as the mRNA transcript (except T nucleobases in the DNA sequence are substituted with U nucleobases in RNA sequence).
A nucleic acid that is complementary to the RNA transcribed from the DNA is termed an “anti-sense” oligonucleotide because its base sequence is complementary to the gene's messenger RNA (mRNA)—the “sense” sequence. Thus, a coding DNA region having a sense sequence of 5′-AAGGTC-3″ will be transcribed to produce a mRNA having a sense sequence of 5′-AAGGUC-3′ and so an antisense oligomer to that sense sequence will have a sequence of 3′-UUCCAG-5′ if it comprises RNA nucleobases, or 3′-TTCCAG-5′ if the antisense oligomer comprises DNA nucleobases.
Currently, a main focus of antisense therapy involves the use of an oligomer or oligonucleotide, approximately 20 nucleotide/nucleosides in length, synthesized to be complementary to the specific “sense” (5′ to 3′ orientation) DNA or mRNA sequence responsible for expression or translation of a targeted protein.
Once introduced into a cell, the antisense oligonucleotide hybridizes to its corresponding mRNA sequence through Watson-Crick binding, forming a heteroduplex. Once a duplex is formed, translation of the protein coded by the sequence of bound mRNA is inhibited. There are several mechanisms through which the oligonucleotide/mRNA duplex may hinder subsequent translation. The most widely accepted explanation for several different antisense agents involves the degradation of the mRNA in the heteroduplex by the ubiquitous enzyme RNase H. RNase H is attracted to the heteroduplex and cleaves the bound mRNA, while leaving the oligonucleotide sequence intact, allowing the oligonucleotide to continue seeking and binding to corresponding mRNA sequences. Some other accepted explanations of translation inhibition through antisense therapy which may occur separately or in conjunction with RNase H activity include, but are not limited to, the blocking of appropriate ribosome assembly that disables the ribosomal complexes' ability to translate, blocking of RNA splicing, and/or impeding appropriate exportation of mRNA.
An apparently separate means used by organisms to control gene expression by limiting translation is known as gene interference, which involves the deployment of short RNA sequences which “silence” the target gene. These short RNA sequences are known as small interfering RNAs or siRNAs. RNA interference is an ancient genetic process in which targeted genes, the blueprints for producing certain proteins, can be turned off.
An enzyme known as Dicer is known to play a key role in RNA interference. Dicer is a ribonuclease that recognizes double-stranded RNA molecules and cuts them into short dsRNAs about 20-25 nucleotides long, usually with a two-base overhang on the 3′ end. The small dsRNA fragments created by Dicer are then assimilated into a large multiprotein complex which guide the dsRNA molecules to destinations in the cell where they bind to their target mRNA sequence and turn off genes. This complex is known as the RNA-induced silencing complex (RISC). RISC has a catalytic component, argonaute, which is an endonuclease capable of degrading messenger RNA (mRNA) if the mRNA has a sequence complementary to that of the siRNA guide strand. Dicer, RISC and the siRNA gene silencing system is therefore a necessary step in many fundamental biological events, including genome rearrangement, stem-cell differentiation, brain development, and viral defense.
Interestingly, it has been determined that the size of the small dsRNAs—the siRNAs—is a determinant of their function. If the dsRNAs are too big or too small, they don't make it into the RISC complexes and so no gene silencing occurs.
Antisense oligomers differ from siRNAs in several ways, most importantly, antisense oligomers are single-stranded. They are also synthetic, and typically modified along the phosphate backbone, and often in select positions of the nucleobases.
In the field of antisense therapy, the introduction of chemically modified nucleosides into nucleic acid molecules, particularly into ribonucleic acid molecules (RNA), provides a powerful tool in overcoming potential limitations of in vivo stability and bioavailability inherent to native RNA or DNA molecules that are delivered exogenously. For example, the use of chemically modified nucleic acid molecules can enable a lower dose of a particular nucleic acid molecule for a given therapeutic effect since chemically modified nucleic acid molecules tend to have a longer half-life in serum. Furthermore, certain chemical modifications can improve the bioavailability of nucleic acid molecules by targeting particular cells or tissues and/or improving cellular uptake of the nucleic acid molecule. Therefore, even if the activity of a chemically modified nucleic acid molecule is reduced as compared to a native nucleic acid molecule, for example when compared to an all RNA nucleic acid molecule, the overall activity of the modified nucleic acid molecule can be greater than the native molecule due to improved stability and/or delivery of the molecule.
One useful chemical modification, termed a locked nucleic acid (LNA), introduces a 2′O-4′C-alkylene bridge wherein the alkylene bridge is a C1-6 alkylene bridge, more particularly, a 2′O-4′C-methylene bridge, at one or more RNA or DNA nucleoside moiety. When LNAs are incorporated into antisense RNA or DNA oligomers they have been shown to greatly increase the stability of the antisense RNA or DNA molecule, and thus to greatly increase bioavailability of the antisense RNA or DNA once it is taken up by the host cell. Other useful chemical modifications that can be introduced into the antisense RNA or DNA oligomers to increase stability and bioavailability of the antisense oligomer include phosphorothioate bonds, or phosphotriester bonds, substituted in place of naturally occurring phosphodiester bonds between the individual RNA or DNA nucleotides.